Novel class of peptides disintegrating biological membranes to aid in the characterization of membrane proteins.

Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

Sulfonated polystyrenes: pH and Mg2+-insensitive amphiphilic copolymers for detergent-free membrane protein isolation.

Amphiphilic polymers are increasingly applied in the detergent-free isolation and functional studies of membrane proteins. However, the carboxylate group present in the structure of many popular variants, such as styrene-maleic acid (SMA) copolymers, brings limitations in terms of polymer sensitivity to precipitation at acidic pH or in the presence of divalent metal cations. Herein, we addressed this problem by replacing carboxylate with the more acidic sulfonate groups. To this end, we synthesized a library of amphiphilic poly[styrene-co-(sodium 4-styrene sulfonate)] copolymers (termed SSS), differing in their molecular weight and overall polarity. Using model cell membranes (Jurkat), we identified two copolymer compositions (SSS-L30 and SSS-L36) that solubilized membranes to an extent similar to SMA. Interestingly, the density gradient ultracentrifugation/SDS-PAGE/Western blotting analysis of cell lysates revealed a distribution of studied membrane proteins in the gradient fractions that was different than for SMA-solubilized membranes. Importantly, unlike SMA, the SSS copolymers remained soluble at low pH and in the presence of Mg2+ ions. Additionally, the solubilization of DMPC liposomes by the lead materials was studied by turbidimetry, DLS, SEC, and high-resolution NMR, revealing, for SSS-L36, the formation of stable particles (nanodiscs), facilitated by the direct hydrophobic interaction of the copolymer phenyls with lipid acyl chains.

Composition of raft-like cell membrane microdomains resistant to styrene-maleic acid copolymer (SMA) solubilization.

An advantageous alternative to the use of detergents in biochemical studies on membrane proteins are the recently developed styrene-maleic acid (SMA) amphipathic copolymers. In our recent study [1] we demonstrated that using this approach, most T cell membrane proteins were fully solubilized (presumably in small nanodiscs), while two types of raft proteins, GPI-anchored proteins and Src family kinases, were mostly present in much larger (>250 nm) membrane fragments markedly enriched in typical raft lipids, cholesterol and lipids containing saturated fatty acid residues. In the present study we demonstrate that disintegration of membranes of several other cell types by means of SMA copolymer follows a similar pattern and we provide a detailed proteomic and lipidomic characterization of these SMA-resistant membrane fragments (SRMs).

Tailoring Butyl Methacrylate/Methacrylic Acid Copolymers for the Solubilization of Membrane Proteins: The Influence of Composition and Molecular Weight.

Low-molecular weight (MW) amphiphilic copolymers have been recently introduced as a powerful tool for the detergent-free isolation of cell membrane proteins. Herein, a screening approach is used to identify a new copolymer type for this application. Via a two-step ATRP/acidolysis procedure, a 3 × 3 matrix of well-defined poly[(butyl methacrylate)-co-(methacrylic acid)] copolymers (denoted BMAA) differing in their MW and ratio of hydrophobic (BMA) and hydrophilic (MAA) units is prepared. Subsequently, using the biologically relevant model (T-cell line Jurkat), two compositions of BMAA copolymers are identified that solubilize cell membranes to an extent comparable to the industry standard, styrene-maleic acid copolymer (SMA), while avoiding the potentially problematic phenyl groups. Surprisingly, while only the lowest-MW variant of the BMA/MAA 2:1 composition is effective, all the copolymers of the BMA/MAA 1:1 composition are found to solubilize the model membranes, including the high-MW variant (MW of 14 000). Importantly, the density gradient ultracentrifugation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting experiments reveal that the BMA/MAA 1:1 copolymers disintegrate the Jurkat membranes differently than SMA, as demonstrated by the different distribution patterns of two tested membrane protein markers. This makes the BMAA copolymers a useful tool for studies on membrane microdomains differing in their composition and resistance to membrane-disintegrating polymers.

Regulation of Inflammatory Response by Transmembrane Adaptor Protein LST1.

LST1 is a small adaptor protein expressed in leukocytes of myeloid lineage. Due to the binding to protein tyrosine phosphatases SHP1 and SHP2 it was thought to have negative regulatory function in leukocyte signaling. It was also shown to be involved in cytoskeleton regulation and generation of tunneling nanotubes. LST1 gene is located in MHCIII locus close to many immunologically relevant genes. In addition, its expression increases under inflammatory conditions such as viral infection, rheumatoid arthritis and inflammatory bowel disease and its deficiency was shown to result in slightly increased sensitivity to influenza infection in mice. However, little else is known about its role in the immune system homeostasis and immune response. Here we show that similar to humans, LST1 is expressed in mice in the cells of the myeloid lineage. In vivo, its deficiency results in alterations in multiple leukocyte subset abundance in steady state and under inflammatory conditions. Moreover, LST1-deficient mice show significant level of resistance to dextran sodium sulphate (DSS) induced acute colitis, a model of inflammatory bowel disease. These data demonstrate that LST1 regulates leukocyte abundance in lymphoid organs and inflammatory response in the gut.

Dysregulated NADPH Oxidase Promotes Bone Damage in Murine Model of Autoinflammatory Osteomyelitis.

Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation. Pstpip2cmo mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1ß by neutrophil granulocytes. In this study, we show that in addition to IL-1ß, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase. Pstpip2cmo neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in Pstpip2cmo mice did not affect IL-1ß levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2cmo mice.

Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis.

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6-RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non-haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4-family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l-deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.

Correction to: EVI2B is a C/EBPα target gene required for granulocytic differentiation and functionality of hematopoietic progenitors.

Since publication of this article, the authors identified an error in the acknowledgments section. The original sentence in acknowledgments reads as follows.

The use of styrene-maleic acid copolymer (SMA) for studies on T cell membrane rafts.

An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.

EVI2B is a C/EBPα target gene required for granulocytic differentiation and functionality of hematopoietic progenitors.

Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.

PSTPIP2, a Protein Associated with Autoinflammatory Disease, Interacts with Inhibitory Enzymes SHIP1 and Csk.

Mutations in the adaptor protein PSTPIP2 are the cause of the autoinflammatory disease chronic multifocal osteomyelitis in mice. This disease closely resembles the human disorder chronic recurrent multifocal osteomyelitis, characterized by sterile inflammation of the bones and often associated with inflammation in other organs, such as the skin. The most critical process in the disease's development is the enhanced production of IL-1ß. This excessive IL-1ß is likely produced by neutrophils. In addition, the increased activity of macrophages, osteoclasts, and megakaryocytes has also been described. However, the molecular mechanism of how PSTPIP2 deficiency results in this phenotype is poorly understood. Part of the PSTPIP2 inhibitory function is mediated by protein tyrosine phosphatases from the proline-, glutamic acid-, serine- and threonine-rich (PEST) family, which are known to interact with the central part of this protein, but other regions of PSTPIP2 not required for PEST-family phosphatase binding were also shown to be indispensable for PSTPIP2 function. In this article, we show that PSTPIP2 binds the inhibitory enzymes Csk and SHIP1. The interaction with SHIP1 is of particular importance because it binds to the critical tyrosine residues at the C terminus of PSTPIP2, which is known to be crucial for its PEST-phosphatase-independent inhibitory effects in different cellular systems. We demonstrate that in neutrophils this region is important for the PSTPIP2-mediated suppression of IL-1ß processing and that SHIP1 inhibition results in the enhancement of this processing. We also describe deregulated neutrophil response to multiple activators, including silica, Ab aggregates, and LPS, which is suggestive of a rather generalized hypersensitivity of these cells to various external stimulants.

LST1/A is a myeloid leukocyte-specific transmembrane adaptor protein recruiting protein tyrosine phosphatases SHP-1 and SHP-2 to the plasma membrane.

Transmembrane adaptor proteins are membrane-anchored proteins consisting of a short extracellular part, a transmembrane domain, and a cytoplasmic part with various protein-protein interaction motifs but lacking any enzymatic activity. They participate in the regulation of various signaling pathways by recruiting other proteins to the proximity of cellular membranes where the signaling is often initiated and propagated. In this work, we show that LST1/A, an incompletely characterized protein encoded by MHCIII locus, is a palmitoylated transmembrane adaptor protein. It is expressed specifically in leukocytes of the myeloid lineage, where it localizes to the tetraspanin-enriched microdomains. In addition, it binds SHP-1 and SHP-2 phosphatases in a phosphotyrosine-dependent manner, facilitating their recruitment to the plasma membrane. These data suggest a role for LST1/A in negative regulation of signal propagation.

SCIMP, a transmembrane adaptor protein involved in major histocompatibility complex class II signaling.

Formation of the immunological synapse between an antigen-presenting cell (APC) and a T cell leads to signal generation in both cells involved. In T cells, the lipid raft-associated transmembrane adaptor protein LAT plays a central role. Its phosphorylation is a crucial step in signal propagation, including the calcium response and mitogen-activated protein kinase activation, and largely depends on its association with the SLP76 adaptor protein. Here we report the discovery of a new palmitoylated transmembrane adaptor protein, termed SCIMP. SCIMP is expressed in B cells and other professional APCs and is localized in the immunological synapse due to its association with tetraspanin-enriched microdomains. In B cells, it is constitutively associated with Lyn kinase and becomes tyrosine phosphorylated after major histocompatibility complex type II (MHC-II) stimulation. When phosphorylated, SCIMP binds to the SLP65 adaptor protein and also to the inhibitory kinase Csk. While the association with SLP65 initiates the downstream signaling cascades, Csk binding functions as a negative regulatory loop. The results suggest that SCIMP is involved in signal transduction after MHC-II stimulation and therefore serves as a regulator of antigen presentation and other APC functions.

Btk is a positive regulator in the TREM-1/DAP12 signaling pathway.

The triggering receptor expressed on myeloid cells 1 (TREM-1) has been implicated in the production of proinflammatory cytokines and chemokines during bacterial infection and sepsis. For downstream signal transduction, TREM-1 is coupled to the ITAM-containing adaptor DAP12. Here, we demonstrate that Bruton tyrosine kinase (Btk), a member of the Tec kinases, becomes phosphorylated upon TREM-1 triggering. In U937-derived cell lines, in which expression of Btk was diminished by shRNA-mediated knockdown, phosphorylation of Erk1/2 and PLCγ1 and Ca²âº mobilization were reduced after TREM-1 stimulation. Importantly, TREM-1-induced production of the pro-inflammatory cytokines, TNF-α and IL-8, and up-regulation of activation/differentiation cell surface markers were impaired in Btk knockdown cells. Similar results were obtained upon TREM-1 stimulation of BMDCs of Btk(-/-) mice. The analysis of cells containing Btk mutants revealed that intact membrane localization and a functional kinase domain were required for TREM-1-mediated signaling. Finally, after TREM-1 engagement, TNF-α production by PBMCs was reduced in the majority of patients suffering from X-linked agammaglobulinemia (XLA), a rare hereditary disease caused by mutations in the BTK gene. In conclusion, our data identify Btk as a positive regulator in the ITAM-mediated TREM-1/DAP12 pathway and suggest its implication in inflammatory processes.

Interaction of late apoptotic and necrotic cells with vitronectin.

BACKGROUND: Vitronectin is an abundant plasma glycoprotein identified also as a part of extracellular matrix. Vitronectin is substantially enriched at sites of injured, fibrosing, inflamed, and tumor tissues where it is believed to be involved in wound healing and tissue remodeling. Little is known about the mechanism of vitronectin localization into the damaged tissues. METHODOLOGY/PRINCIPAL FINDINGS: 2E12 antibody has been described to bind a subset of late apoptotic cells. Using immunoisolation followed by mass spectrometry, we identified the antigen recognized by 2E12 antibody as vitronectin. Based on flow cytometry, we described that vitronectin binds to the late apoptotic and necrotic cells in cell cultures in vitro as well as in murine thymus and spleen in vivo. Confocal microscopy revealed that vitronectin binds to an intracellular cytoplasmic structure after the membrane rupture. CONCLUSIONS/SIGNIFICANCE: We propose that vitronectin could serve as a marker of membrane disruption in necrosis and apoptosis for flow cytometry analysis. Moreover, we suggest that vitronectin binding to dead cells may represent one of the mechanisms of vitronectin incorporation into the injured tissues.

Regulation of Src family kinases involved in T cell receptor signaling by protein-tyrosine phosphatase CD148.

CD148 is a receptor-like protein-tyrosine phosphatase known to inhibit transduction of mitogenic signals in non-hematopoietic cells. Similarly, in the hematopoietic lineage, CD148 inhibited signal transduction downstream of T cell receptor. However, it also augmented immunoreceptor signaling in B cells and macrophages via dephosphorylating C-terminal tyrosine of Src family kinases (SFK). Accordingly, endogenous CD148 compensated for the loss of the main SFK activator CD45 in murine B cells and macrophages but not in T cells. Hypothetical explanations for the difference between T cells and other leukocyte lineages include the inability of CD148 to dephosphorylate a specific set of SFKs involved in T cell activation or the lack of CD148 expression during critical stages of T cell development. Here we describe striking differences in CD148 expression between human and murine thymocyte subsets, the only unifying feature being the absence of CD148 during the positive selection when the major developmental block occurs under CD45 deficiency. Moreover, we demonstrate that similar to CD45, CD148 has both activating and inhibitory effects on the SFKs involved in TCR signaling. However, in the absence of CD45, activating effects prevail, resulting in functional complementation of CD45 deficiency in human T cell lines. Importantly, this is independent of the tyrosines in the CD148 C-terminal tail, contradicting the recently proposed phosphotyrosine displacement model as a mechanism of SFK activation by CD148. Collectively, our data suggest that differential effects of CD148 in T cells and other leukocyte subsets cannot be explained by the CD148 inability to activate T cell SFKs but rather by its dual inhibitory/activatory function and specific expression pattern.

PRR7 is a transmembrane adaptor protein expressed in activated T cells involved in regulation of T cell receptor signaling and apoptosis.

Transmembrane adaptor proteins (TRAPs) are important organizers and regulators of immunoreceptor-mediated signaling. A bioinformatic search revealed several potential novel TRAPs, including a highly conserved protein, proline rich 7 (PRR7), previously described as a component of the PSD-95/N-methyl-d-aspartate receptor protein complex in postsynaptic densities (PSD) of rat neurons. Our data demonstrate that PRR7 is weakly expressed in other tissues but is readily up-regulated in activated human peripheral blood lymphocytes. Transient overexpression of PRR7 in Jurkat T cell line led to gradual apoptotic death dependent on the WW domain binding motif surrounding Tyr-166 in the intracellular part of PRR7. To circumvent the pro-apoptotic effect of PRR7, we generated Jurkat clones with inducible expression of PRR7 (J-iPRR7). In these cells acute induction of PRR7 expression had a dual effect. It resulted in up-regulation of the transcription factor c-Jun and the activation marker CD69 as well as enhanced production of IL-2 after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. On the other hand, expression of PRR7 inhibited general tyrosine phosphorylation and calcium influx after T cell receptor cross-linking by antibodies. Moreover, we found PRR7 constitutively tyrosine-phosphorylated and associated with Src. Collectively, these data indicate that PRR7 is a potential regulator of signaling and apoptosis in activated T cells.

High expression of cytoskeletal protein drebrin in TEL/AML1pos B-cell precursor acute lymphoblastic leukemia identified by a novel monoclonal antibody.

The expression of drebrin, a cytoskeletal protein newly estimated by expression profiling to correlate with the genotype and prognosis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), was examined by independent methods. After demonstrating its higher expression in TEL/AML1(pos) BCP-ALL by quantitative reverse transcriptase polymerase chain reaction, we developed an anti-drebrin monoclonal antibody (mAb). In a cohort of 86 children with BCP-ALL, we found increased expression of drebrin in TEL/AML1(pos) ALL. In conclusion, relationship of drebrin expression and prognosis or genotype can now be assessed using flow cytometry.

The effects of membrane compartmentalization of csk on TCR signaling.

The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described "heavy rafts" or non-raft membrane. Our data show that only Csk targeted into "classical" raft but not to "heavy raft" or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.